Project Summary/Abstract The study of antiviral defenses in bacteria has led to the discovery and practical use of valuable tools such as restriction enzymes and CRISPR-Cas. However, the diversity of bacteriophage defense strategies is not fully appreciated and limited efforts have been made to discover and characterize novel systems. Sequence-based analyses suggest that there is an abundance of uncharacterized bacteriophage-defense genes that have yet to be identified or experimentally validated. Importantly, phage resistance, like antibiotic resistance, may disseminate to human pathogens under selection, posing a potential barrier to the practical use of phage therapy as an antibiotic alternative. Therefore, it is important to have a more complete understanding of the diversity of phage defense systems and their potential to horizontally transfer. This study proposes a high-throughput functional selection approach to identify phage-defense loci in human microbial metagenomes and strain collections. Shotgun genomic libraries will be expressed in Escherichia coli and selected for acquired resistance to several types of E. coli bacteriophage. The proposed strategy uses a sensitive selection method that detects full or partial resistance and is coupled to deep sequencing post-selection to increase throughput. Preliminary experiments have revealed that small-insert (~2 kb) human metagenomic libraries contain hundreds of clones that confer bacteriophage T4 resistance to E. coli. In this data, novel defense systems were discovered such as a single-gene system that belongs to a conserved family of unknown function and has not been studied to date. Originating from Neisseria, this gene protects E. coli from T4, supporting the hypothesis that the human microflora harbors a mobile pool of phage resistance. This method is poised to immediately uncover new information about a fundamental aspect of bacterial biology. In addition, it will serve as a model to determine the dissemination potential of bacteriophage resistance genes. Finally, this study aims to take genetic and biochemical approaches to investigate the molecular mechanisms of novel defense systems in detail, providing new insights into how bacteria sense and respond to their viruses. In all, this study will have long-term impacts on our basic understanding of bacterial antiviral immunity and inform bacteriophage therapy moving forward. In addition to research, this training plan will incorporate scientific communication during weekly meetings and conferences, teaching and mentorship opportunities, and professional development. Training will take place at Massachusetts Institute of Technology in a productive, high-quality research lab, and will provide the resources required for the successful completion of all aspects of the training.